Flaviviruses and alphaviruses are spread by mosquito and tick vectors and cause severe human and animal illnesses such as encephalitis and hemorrhagic fever. These viruses include potential bioterrorist agents that are category A-C pathogens, such as the flavivirus dengue virus (DV) and the alphavirus Chikungunya virus (CV). DV is of particular concern as it has dramatically reemerged to become endemic in more than 100 countries including the US, and is now a global health problem. Alphaviruses can also be important emerging pathogens, as exemplified by the recent outbreak of CV infection with several million cases reported to date in India. Antiviral strategies for the flaviviruses and alphaviruses are urgently needed. The flavivirus and alphavirus membrane fusion proteins are members of the class II virus membrane fusion proteins. They are structurally very similar and refold to a homotrimer form to mediate virus fusion and infection. In collaboration with Dr. Felix Rey, we determined the structure of the homotrimer conformation of the fusion protein from the alphavirus Semliki Forest virus (SFV). The SFV homotrimer structure is strikingly similar to that of DV. Using the structures as a guide, we have recently developed recombinant forms of domain III (DUN) from the SFV and DV fusion proteins. These recombinant Dill proteins act as specific dominant-negative inhibitors of virus fusion and infection, and are the basis for our proposed screen for small molecule inhibitors of DV and CV. In aim 1 we will use our recombinant protein systems to define the mechanism of DV and CV fusion protein trimerization and the steps in trimer formation. In aim 2, we will develop fluorescence-based in vitro assays for the binding of DIM to the trimer core of DV and CV. In aim 3 we will adapt this assay to a high throughput format. We will then use the assay in high throughput screens of small molecule libraries available through the NIH and the Northeast Biodefense Consortium, and validate hits by tests of virus fusion and infection. Ultimately, such inhibitors will be lead compounds for antiviral therapy, and important research tools to understand the class II virus fusion reaction.